Molds (i.e., toxigenic and other septate molds) are ubiquitous in the environment. Mold is the common name for various types of fungi. Molds are usually found in moist, warm environments. Because molds grow in wet or moist indoor environments, people are exposed to molds or their byproducts through either direct contact, or through the air, if molds or mold byproducts are aerosolized. Exposure to molds can cause a number of adverse effects including allergic reactions, asthma attacks, and infections, particularly in individuals with immune system deficiencies.
Adverse effects from molds may occur when individuals are exposed to large doses of chemicals, known as mycotoxins, which are fungal metabolites (Samson et al., 1985; Burge, 1990; Flannigan et al., 1991). Mycotoxins have toxic effects ranging from severe irritations, such as allergic reactions and asthma, to immuno-suppression and cancer. Most mycotoxins are cytotoxic and exert their effects by interfering with vital cellular processes such as protein, RNA, and DNA synthesis. As a result, mycotoxins may be damaging to the skin, the lungs, the gut, and the like. The combined outcome may increase the susceptibility of the exposed individual to infectious diseases and, possibly, to cancer. Almost all of the studies to date focus on disease induced by mycotoxins ingested in contaminated food (Baxter et al., 1981), but mycotoxins are secondary metabolites of fungal spores and can enter the body through the respiratory tract.
In heavily contaminated environments, neurotoxic symptoms related to airborne mycotoxin exposure have been reported (Croft et al., 1986). Skin is another potential route of exposure to the mycotoxins of several fungi which have caused cases of severe dermatosis (Vennewald and Wollina, 2005). These same molds may cause invasive mold infection among patients with diseases which render the patient immuno-suppressed such as leukemia, lymphoma, and many cancers (Kontoyiannis, D P et al, 2005). The mold infections in such patients are often fatal with a documented fatally rate of 92% (Paterson and Singh, 1999).
Aspergillus terreus is a species of fungus that is widespread throughout the world and may be found in warm arable soils. Aspergillus terreus is known to cause opportunistic infections in patients, particularly patients with deficient immune systems. For example, patients who have received a transplant are susceptible to infection caused by Aspergillus terreus. Importantly, infection with Aspergillus terreus is more likely to result in invasive, disseminated disease than infection with other Aspergillus species.
A definitive and early diagnosis of a fungal infection, such as a fungal infection caused by Aspergillus terreus, is crucial for patient treatment and management. A diagnosis of a fungal infection is often rendered late in the disease process, often even as late as autopsy (Kontoyiannis et al, 2000; Vogeser et al., 1997). The reasons for the late diagnosis of fungal infections include the lack of good clinical specimens, the difficulty in differentiating invasive mold infections from other types of infections, the lack of identification of molds with special stains in pathological specimens (i.e., these assays have a high error rate, a low sensitivity, and low specificity), and the lack of an ability to obtain an antibody-based diagnosis in immuno-compromised patients.
Aspergillus niger is a common species of fungus that is ubiquitous in soil and is also commonly found in indoor environments. Aspergillus niger is a common contaminant of various foods, for example fruits such as grapes and vegetables such as onions, as well as peanuts. Furthermore, Aspergillus niger is known to cause infections in patients, for example aspergillosis (a serious lung disease) and otomycosis (a fungal ear infection), as well as infections occurring in the endocardium, kidneys, respiratory tract, and digestive tract.
Thus, a reliable, sensitive, specific, and rapid method for Aspergillus terreus detection and Aspergillus niger detection in patient body fluids and tissues is needed. Applicant's present invention is based on the development of a reliable, sensitive, specific, and rapid method for detecting Aspergillus terreus DNA and Aspergillus niger DNA in patient body fluids and tissues. Furthermore, the method for detecting Aspergillus terreus DNA in patient body fluids and tissues can be combined with detection of an Aspergillus terreus mycotoxin. If Aspergillus terreus DNA or mycotoxins can be identified in patient tissue or body fluids, the identification may serve as a potential diagnostic method 1) to identify patients at risk for developing disease states related to Aspergillus terreus infections, or 2) to rapidly determine the cause of diseases related to Aspergillus terreus infections so that effective treatment regimens can be developed for patients exposed to molds and experiencing symptoms resulting from mold infection. The same applies to detection of Aspergillus niger DNA and mycotoxins.
The present invention provides methods for detecting and identifying, in patient tissue and patient body fluid specimens, 1) Aspergillus terreus DNA from fungal spores, 2) mycotoxins produced by Aspergillus terreus, 3) Aspergillus niger DNA from fungal spores, and 4) mycotoxins produced by Aspergillus niger. The present invention provides reliable, sensitive, and specific diagnostic tests for the presence of Aspergillus, Aspergillus niger, Aspergillus terreus fungal toxins, and Aspergillus niger fungal toxins in patient tissue and body fluids. The Applicant has developed Aspergillus terreus mycotoxin, Aspergillus terreus DNA, Aspergillus niger mycotoxin, and Aspergillus niger DNA extraction procedures and has supplemented those methods by developing detection methods. The detection methods employ antibody-based identification for mycotoxins and, for Aspergillus terreus DNA or Aspergillus niger DNA, the use of amplification of DNA with primers that specifically and selectively amplify Aspergillus terreus DNA or Aspergillus niger DNA isolated from patient tissues and body fluids.
The following embodiments are contemplated and are non-limiting:
1. A method of identifying an Aspergillus terreus fungal species in a patient tissue or a patient body fluid, the method comprising the steps of:                extracting and recovering DNA of the Aspergillus terreus fungal species from the patient tissue or the patient body fluid;        amplifying the DNA;        hybridizing a probe to the DNA to specifically identify the fungal species, wherein the probe has a sequence consisting of SEQ ID NO: 1; and        specifically identifying the Aspergillus terreus fungal species.        
2. The method of clause 1, wherein the amplifying step is performed with primers that hybridize to the DNA.
3. The method of clause 1 or clause 2, further comprising using a forward primer and a reverse primer to amplify the DNA, wherein the forward primer consists of a sequence of SEQ ID NO: 2 and the reverse primer consists of a sequence of SEQ ID NO: 3.
4. The method of any one of clauses 1 to 3, wherein the DNA is amplified using PCR.
5. The method of clause 4, wherein the PCR is real-time PCR.
6. The method of any one of clauses 1 to 5, wherein the probe is fluorescently labeled.
7. The method of any one of clauses 1 to 6, wherein the probe is bound to a bead dyed with a fluorochrome.
8. The method of any one of clauses 1 to 7, wherein the amplified DNA is internal transcribed spacer regions of nuclear ribosomal DNA.
9. The method of any one of clauses 1 to 8, wherein the body fluid is selected from the group consisting of urine, nasal secretions, nasal washes, bronchial lavages, bronchial washes, spinal fluid, sputum, gastric secretions, seminal fluid, other reproductive tract secretions, lymph fluid, whole blood, serum, and plasma.
10. The method of any one of clauses 1 to 9, wherein the method further comprises the step of administering an anti-fungal agent to the patient.
11. The method of any one of clauses 1 to 10, wherein the method further comprises the step of testing for the presence of an Aspergillus terreus mycotoxin in the patient tissue or the patient body fluid prior to extraction and recovery of the DNA.
12. The method of any one of clauses 1 to 11, wherein the patient is a transplant patient.
13. A method of identifying a patient at risk for an Aspergillus terreus fungal infection, the method comprising the steps of:                extracting and recovering DNA of the Aspergillus terreus fungal species from a tissue or a body fluid of the patient;        amplifying the DNA;        hybridizing a probe to the DNA to specifically identify the Aspergillus terreus fungal species, wherein the probe consists of a sequence of SEQ ID NO: 1; and        specifically identifying the Aspergillus terreus fungal species.        
14. The method of clause 13, wherein the body fluid is selected from the group consisting of urine, nasal secretions, nasal washes, bronchial lavages, bronchial washes, spinal fluid, sputum, gastric secretions, seminal fluid, other reproductive tract secretions, lymph fluid, whole blood, serum, and plasma.
15. The method of clause 13 or clause 14, wherein the probe is bound to a bead dyed with a fluorochrome.
16. The method of any one of clauses 13 to 15, wherein the method further comprises the step of developing an effective treatment regimen for the patient.
17. The method of any one of clauses 13 to 16, wherein the method further comprises the step of administering an anti-fungal agent to the patient.
18. The method of any one of clauses 13 to 17, wherein the method further comprises the step of testing for the presence of an Aspergillus terreus mycotoxin in the tissue or the body fluid prior to extraction and recovery of the DNA.
19. The method of clause 18, wherein the testing for the presence of the Aspergillus terreus mycotoxin comprises contacting the mycotoxin with an antibody directed against the mycotoxin.
20. The method of any one of clauses 13 to 19, wherein the patient is a transplant patient.
21. A method of identifying a patient with an Aspergillus terreus fungal infection, the method comprising the steps of:                extracting and recovering DNA of the Aspergillus terreus fungal species from a tissue or a body fluid of the patient;        amplifying the DNA;        hybridizing a probe to the DNA to specifically identify the Aspergillus terreus fungal species, wherein the probe consists of a sequence of SEQ ID NO: 1; and        specifically identifying the Aspergillus terreus fungal species.        
22. The method of clause 21, wherein the body fluid is selected from the group consisting of urine, nasal secretions, nasal washes, bronchial lavages, bronchial washes, spinal fluid, sputum, gastric secretions, seminal fluid, other reproductive tract secretions, lymph fluid, whole blood, serum, and plasma.
23. The method of clause 21 or clause 22, wherein the probe is bound to a bead dyed with a fluorochrome.
24. The method of any one of clauses 21 to 23, wherein the method further comprises the step of developing an effective treatment regimen for the patient.
25. The method of any one of clauses 21 to 24, wherein the method further comprises the step of administering an anti-fungal agent to the patient.
26. The method of any one of clauses 21 to 25, wherein the method further comprises the step of testing for the presence of an Aspergillus terreus mycotoxin in the tissue or the body fluid prior to extraction and recovery of the DNA.
27. The method of clause 26, wherein the testing for the presence of the Aspergillus terreus mycotoxin comprises contacting the mycotoxin with an antibody directed against the mycotoxin.
28. The method of any one of clauses 21 to 27, wherein the patient is a transplant patient.
29. An isolated purified nucleic acid comprising SEQ ID NO: 1 or a sequence that hybridizes under highly stringent conditions to a sequence comprising SEQ ID NO: 1.
30. An isolated, purified nucleic acid consisting of SEQ ID NO: 1 or a sequence that hybridizes under highly stringent conditions to a sequence consisting of SEQ ID NO: 1.
31. An isolated, purified nucleic acid comprising SEQ ID NO: 2 or a sequence that hybridizes under highly stringent conditions to a sequence comprising SEQ ID NO: 2.
32. An isolated, purified nucleic acid consisting of SEQ ID NO: 2 or a sequence that hybridizes under highly stringent conditions to a sequence consisting of SEQ ID NO: 2.
33. An isolated, purified nucleic acid comprising SEQ ID NO: 3 or a sequence that hybridizes under highly stringent conditions to a sequence comprising SEQ ID NO: 3.
34. An isolated, purified nucleic acid consisting of SEQ ID NO: 3 or a sequence that hybridizes under highly stringent conditions to a sequence consisting of SEQ ID NO: 3.
35. A kit comprising an isolated, purified nucleic acid with a sequence comprising SEQ ID NO: 1.
36. A kit comprising an isolated, purified nucleic acid with a sequence consisting of SEQ ID NO: 1.
37. The kit of clause 35 or clause 36 further comprising a purified nucleic acid with a sequence comprising SEQ ID NO: 2 and a purified nucleic acid with a sequence comprising SEQ ID NO: 3.
38. The kit of clause 35 or clause 36 further comprising an isolated, purified nucleic acid with a sequence consisting of SEQ ID NO: 2 and an isolated, purified nucleic acid with a sequence consisting of SEQ ID NO: 3.
39. The kit of any one of clauses 35 to 38 further comprising components for the extraction and recovery of an Aspergillus terreus mycotoxin from a body fluid or a tissue of a patient and components for identification of the mycotoxin.
40. The kit of clause 39 wherein the components for identification of the mycotoxin include beads dyed with a fluorochrome and coupled to antibodies to the mycotoxin or to the mycotoxin or to a mycotoxin antigen.
41. The kit of clause 39 wherein the components for identification of the mycotoxin comprise an antibody directed against the mycotoxin.
42. A method of identifying an Aspergillus niger fungal species in a patient tissue or a patient body fluid, the method comprising the steps of:                extracting and recovering DNA of the Aspergillus niger fungal species from the patient tissue or the patient body fluid;        amplifying the DNA;        hybridizing a probe to the DNA to specifically identify the fungal species, wherein the probe has a sequence consisting of SEQ ID NO: 4; and        specifically identifying the Aspergillus niger fungal species.        
43. The method of clause 42, wherein the amplifying step is performed with primers that hybridize to the DNA.
44. The method of clause 42 or clause 43, further comprising using a forward primer and a reverse primer to amplify the DNA, wherein the forward primer consists of a sequence of SEQ ID NO: 5 and the reverse primer consists of a sequence of SEQ ID NO: 6.
45. The method of any one of clauses 42 to 44, wherein the DNA is amplified using PCR.
46. The method of clause 45, wherein the PCR is real-time PCR.
47. The method of any one of clauses 42 to 46, wherein the probe is fluorescently labeled.
48. The method of any one of clauses 42 to 47, wherein the probe is bound to a bead dyed with a fluorochrome.
49. The method of any one of clauses 42 to 48, wherein the amplified DNA is internal transcribed spacer regions of nuclear ribosomal DNA.
50. The method of any one of clauses 42 to 49, wherein the body fluid is selected from the group consisting of urine, nasal secretions, nasal washes, bronchial lavages, bronchial washes, spinal fluid, sputum, gastric secretions, seminal fluid, other reproductive tract secretions, lymph fluid, whole blood, serum, and plasma.
51. The method of any one of clauses 42 to 50, wherein the method further comprises the step of administering an anti-fungal agent to the patient.
52. The method of any one of clauses 42 to 51, wherein the method further comprises the step of testing for the presence of an Aspergillus niger mycotoxin in the patient tissue or the patient body fluid prior to extraction and recovery of the DNA.
53. The method of any one of clauses 42 to 52, wherein the patient is a transplant patient.
54. A method of identifying a patient at risk for an Aspergillus niger fungal infection, the method comprising the steps of:                extracting and recovering DNA of the Aspergillus niger fungal species from a tissue or a body fluid of the patient;        amplifying the DNA;        hybridizing a probe to the DNA to specifically identify the Aspergillus niger fungal species, wherein the probe consists of a sequence of SEQ ID NO: 4; and        specifically identifying the Aspergillus niger fungal species.        
55. The method of clause 54, wherein the body fluid is selected from the group consisting of urine, nasal secretions, nasal washes, bronchial lavages, bronchial washes, spinal fluid, sputum, gastric secretions, seminal fluid, other reproductive tract secretions, lymph fluid, whole blood, serum, and plasma.
56. The method of clause 54 or clause 55, wherein the probe is bound to a bead dyed with a fluorochrome.
57. The method of any one of clauses 54 to 56, wherein the method further comprises the step of developing an effective treatment regimen for the patient.
58. The method of any one of clauses 54 to 57, wherein the method further comprises the step of administering an anti-fungal agent to the patient.
59. The method of any one of clauses 54 to 58, wherein the method further comprises the step of testing for the presence of an Aspergillus niger mycotoxin in the tissue or the body fluid prior to extraction and recovery of the DNA.
60. The method of clause 59, wherein the testing for the presence of the Aspergillus niger mycotoxin comprises contacting the mycotoxin with an antibody directed against the mycotoxin.
61. The method of any one of clauses 54 to 60, wherein the patient is a transplant patient.
62. A method of identifying a patient with an Aspergillus niger fungal infection, the method comprising the steps of:                extracting and recovering DNA of the Aspergillus niger fungal species from a tissue or a body fluid of the patient;        amplifying the DNA;        hybridizing a probe to the DNA to specifically identify the Aspergillus niger fungal species, wherein the probe consists of a sequence of SEQ ID NO: 4; and        specifically identifying the Aspergillus niger fungal species.        
63. The method of clause 62, wherein the body fluid is selected from the group consisting of urine, nasal secretions, nasal washes, bronchial lavages, bronchial washes, spinal fluid, sputum, gastric secretions, seminal fluid, other reproductive tract secretions, lymph fluid, whole blood, serum, and plasma.
64. The method of clause 62 or clause 63, wherein the probe is bound to a bead dyed with a fluorochrome.
65. The method of any one of clauses 62 to 64, wherein the method further comprises the step of developing an effective treatment regimen for the patient.
66. The method of any one of clauses 62 to 65, wherein the method further comprises the step of administering an anti-fungal agent to the patient.
67. The method of any one of clauses 62 to 66, wherein the method further comprises the step of testing for the presence of an Aspergillus niger mycotoxin in the tissue or the body fluid prior to extraction and recovery of the DNA.
68. The method of clause 67, wherein the testing for the presence of the Aspergillus niger mycotoxin comprises contacting the mycotoxin with an antibody directed against the mycotoxin.
69. The method of any one of clauses 62 to 68, wherein the patient is a transplant patient.
70. An isolated purified nucleic acid comprising SEQ ID NO: 4 or a sequence that hybridizes under highly stringent conditions to a sequence comprising SEQ ID NO: 4.
71. An isolated, purified nucleic acid consisting of SEQ ID NO: 4 or a sequence that hybridizes under highly stringent conditions to a sequence consisting of SEQ ID NO: 4.
72. A kit comprising an isolated, purified nucleic acid with a sequence comprising SEQ ID NO: 4.
73. A kit comprising an isolated, purified nucleic acid with a sequence consisting of SEQ ID NO: 4.
74. The kit of clause 72 or clause 73 further comprising a purified nucleic acid with a sequence comprising SEQ ID NO: 5 and a purified nucleic acid with a sequence comprising SEQ ID NO: 6.
75. The kit of clause 72 or clause 73 further comprising an isolated, purified nucleic acid with a sequence consisting of SEQ ID NO: 5 and an isolated, purified nucleic acid with a sequence consisting of SEQ ID NO: 6.
76. The kit of any one of clauses 72 to 75 further comprising components for the extraction and recovery of an Aspergillus niger mycotoxin from a body fluid or a tissue of a patient and components for identification of the mycotoxin.
77. The kit of clause 76 wherein the components for identification of the mycotoxin include beads dyed with a fluorochrome and coupled to antibodies to the mycotoxin or to the mycotoxin or to a mycotoxin antigen.
78. The kit of clause 76 wherein the components for identification of the mycotoxin comprise an antibody directed against the mycotoxin.
In any of the above-described method embodiments for Aspergillus terreus, the probe can consist of SEQ ID NO: 1. In any of the above-described method embodiments for Aspergillus terreus, the primers can consist of SEQ ID NO: 2 or SEQ ID NO: 3.
In any of the above-described method embodiments for Aspergillus niger, the probe can consist of SEQ ID NO: 4. In any of the above-described method embodiments for Aspergillus niger, the primers can consist of SEQ ID NO: 5 or SEQ ID NO: 6.
In one illustrative embodiment, a method is provided of identifying an Aspergillus terreus fungal species in a patient tissue or a patient body fluid. The method comprises the steps of extracting and recovering DNA of the Aspergillus terreus fungal species from the patient tissue or the patient body fluid, amplifying the DNA, hybridizing a probe to the DNA to specifically identify the fungal species, wherein the probe has a sequence consisting of SEQ ID NO: 1, and specifically identifying the Aspergillus terreus fungal species.
In another embodiment, a method is provided of identifying a patient at risk for an Aspergillus terreus fungal infection. The method comprises the steps of extracting and recovering DNA of the Aspergillus terreus fungal species from a tissue or a body fluid of the patient, amplifying the DNA, hybridizing a probe to the DNA to specifically identify the Aspergillus terreus fungal species, wherein the probe consists of a sequence of SEQ ID NO: 1, and specifically identifying the Aspergillus terreus fungal species.
In yet another embodiment, a method is provided of identifying a patient with an Aspergillus terreus fungal infection. The method comprises the steps of extracting and recovering DNA of the Aspergillus terreus fungal species from a tissue or a body fluid of the patient, amplifying the DNA, hybridizing a probe to the DNA to specifically identify the Aspergillus terreus fungal species, wherein the probe consists of a sequence of SEQ ID NO: 1, and specifically identifying the Aspergillus terreus fungal species.
In another illustrative embodiment, an isolated purified nucleic acid is provided. In one embodiment, the nucleic acid comprises SEQ ID NO: 1 or a sequence that hybridizes under highly stringent conditions to a sequence comprising SEQ ID NO: 1. In another embodiment, the nucleic acid consists of SEQ ID NO: 1 or a sequence that hybridizes under highly stringent conditions to a sequence consisting of SEQ ID NO: 1. In yet another embodiment, the nucleic acid comprises SEQ ID NO: 2 or a sequence that hybridizes under highly stringent conditions to a sequence comprising SEQ ID NO: 2. In another embodiment, the nucleic acid consists of SEQ ID NO: 2 or a sequence that hybridizes under highly stringent conditions to a sequence consisting of SEQ ID NO: 2. In yet another embodiment, the nucleic acid comprises SEQ ID NO: 3 or a sequence that hybridizes under highly stringent conditions to a sequence comprising SEQ ID NO: 3. In another embodiment, the nucleic acid consists of SEQ ID NO: 3 or a sequence that hybridizes under highly stringent conditions to a sequence consisting of SEQ ID NO: 3.
In another embodiment, a kit is provided. The kit can comprise an isolated, purified nucleic acid with a sequence comprising SEQ ID NO: 1, or an isolated, purified nucleic acid with a sequence consisting of SEQ ID NO: 1. The kit can also comprise components for the extraction and recovery of DNA or an Aspergillus terreus mycotoxin and components for DNA amplification and instructions for use of the kit.
In one illustrative embodiment, a method is provided of identifying an Aspergillus niger fungal species in a patient tissue or a patient body fluid. The method comprises the steps of extracting and recovering DNA of the Aspergillus niger fungal species from the patient tissue or the patient body fluid, amplifying the DNA, hybridizing a probe to the DNA to specifically identify the fungal species, wherein the probe has a sequence consisting of SEQ ID NO: 4, and specifically identifying the Aspergillus niger fungal species.
In another embodiment, a method is provided of identifying a patient at risk for an Aspergillus niger fungal infection. The method comprises the steps of extracting and recovering DNA of the Aspergillus niger fungal species from a tissue or a body fluid of the patient, amplifying the DNA, hybridizing a probe to the DNA to specifically identify the Aspergillus niger fungal species, wherein the probe consists of a sequence of SEQ ID NO: 4, and specifically identifying the Aspergillus niger fungal species.
In yet another embodiment, a method is provided of identifying a patient with an Aspergillus niger fungal infection. The method comprises the steps of extracting and recovering DNA of the Aspergillus niger fungal species from a tissue or a body fluid of the patient, amplifying the DNA, hybridizing a probe to the DNA to specifically identify the Aspergillus niger fungal species, wherein the probe consists of a sequence of SEQ ID NO: 4, and specifically identifying the Aspergillus niger fungal species.
In another illustrative embodiment, an isolated purified nucleic acid is provided. In one embodiment, the nucleic acid comprises SEQ ID NO: 4 or a sequence that hybridizes under highly stringent conditions to a sequence comprising SEQ ID NO: 4. In another embodiment, the nucleic acid consists of SEQ ID NO: 4 or a sequence that hybridizes under highly stringent conditions to a sequence consisting of SEQ ID NO: 4. In yet another embodiment, the nucleic acid comprises SEQ ID NO: 5 or a sequence that hybridizes under highly stringent conditions to a sequence comprising SEQ ID NO: 5. In another embodiment, the nucleic acid consists of SEQ ID NO: 5 or a sequence that hybridizes under highly stringent conditions to a sequence consisting of SEQ ID NO: 5. In yet another embodiment, the nucleic acid comprises SEQ ID NO: 6 or a sequence that hybridizes under highly stringent conditions to a sequence comprising SEQ ID NO: 6. In another embodiment, the nucleic acid consists of SEQ ID NO: 6 or a sequence that hybridizes under highly stringent conditions to a sequence consisting of SEQ ID NO: 6.
In another embodiment, a kit is provided. The kit can comprise an isolated, purified nucleic acid with a sequence comprising SEQ ID NO: 4, or an isolated, purified nucleic acid with a sequence consisting of SEQ ID NO: 4. The kit can also comprise components for the extraction and recovery of DNA or an Aspergillus niger mycotoxin and components for DNA amplification and instructions for use of the kit.